The ultimate goal of the research proposed here is to elucidate the molecular mechanism of the H+-translocating ATPase, the enzyme which catalyzes ATP synthesis in energy transducing membranes which is driven by electron transport. To achieve this end chemical modification studies are being carried out which modify amino acid side chains in the bovine mitochondiral F1-ATPase with radioactive reagents which inactivate the enzyme. Essential residues appear to be located in three distinct sites, the catalytic site which resides in the Beta subunit, a regulatory site in the Alpha subunit which responds to adenine nucleotides, and a third site which also resides in the Beta subunit which appears to be involved in the propagation of information from the regulatory site to the catalytic site. Amino acid sequence analysis around the residues which are labeled by the radioactive reagents is being used to identify the amino acid residues which have a functional role at each of these sites. Based on previous results, a molecular mechanism is proposed for ATP synthesis catalyzed by the H+-translocating ATPase. It is proposed that a tyrosine residue in the Beta subunit which is labeled by the ATP affinity analogue, 5'-p-F-sulfonylbenzoyladenosine, functions as a general acid during ATP synthesis. This residue is suggested to be reprotonated during catalysis by a trans-membrane proton relay system which shuttles protons, translocated by electron transport, to the active site of the F1-ATPase from the opposite side of the membrane. The purpose of the experiments proposed here is to test the validity of this hypothesis by providing a more complete understanding of the location and function of the various amino acid side chains in the F1-ATPase which are essential for activity. The results of these experiments will provide important structural information which will be necessary to elucidate the molecular mechanism of the H+-translocating ATPase, no matter what it might turn out to be. The elucidation of this mechanism will have great bearing on our understanding of transport processes in membranes in general.